ABSTRACT
Protein palmitoylation or S-acylation has emerged as a key regulator of cellular processes. Increasing evidence shows that this modification is not restricted to palmitate but it can include additional fatty acids, raising the possibility that differential S-acylation contributes to the fine-tuning of protein activity. However, methods to profile the acyl moieties attached to proteins are scarce. Herein, we report a method for the identification and quantification of lipids bound to proteins that relies on hydroxylamine treatment and mass spectrometry analysis of fatty acid hydroxamates. This method has enabled unprecedented and extensive profiling of the S-acylome in different cell lines and tissues and has shed light on the substrate specificity of some S-acylating enzymes. Moreover, we could extend it to quantify also the acyl-CoAs, which are thioesters formed between a fatty acid and a coenzyme A, overcoming many of the previously described challenges for the detection of such species. Importantly, the simultaneous analysis of the lipid fraction and the proteome allowed us to establish, for the first time, a direct correlation between the endogenous levels of acyl-CoAs and the S-acylation profile of its proteome.
ABSTRACT
Ras proteins require membrane association for proper function. This process is tightly regulated by reversible palmitoylation that controls not only the distribution over different subcellular compartments but also Ras compartmentalization within membrane subdomains. As a result, there is a growing interest in protein palmitoylation and the enzymes that control this process. In this minireview, we discuss how palmitoylation affects the localization and function of Ras proteins. A better understanding of the regulatory mechanism controlling protein lipidation is expected to provide new insights into the functional role of these modifications and may ultimately lead to the development of novel therapeutic approaches.